Method for microscopy, and microscope

ABSTRACT

A method for microscopy includes generating pulsed illuminating light including wavelengths in a spectral region. A detection spectral region within the spectral region is defined. Using a dynamically controllable mask, light components of the illuminating light that comprise wavelengths within the detection spectral region are influenced. A specimen is illuminated with the influenced illuminating light. Detection light proceeding from the specimen within the detection spectral region is detected.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 10/601,804, filed Jun.23, 2003, which claims priority to German patent application 102 28374.5. The subject matter of both of these applications is herebyincorporated by reference herein.

FIELD OF THE INVENTION

The invention concerns a method for microscopy. The inventionfurthermore concerns a microscope and a confocal scanning microscope.

BACKGROUND OF THE INVENTION

For the investigation of biological specimens, it has been usual forsome time to prepare the specimen using optical markers, in particularfluorescent dyes. Often, for example in the field of genetic research,several different fluorescent dyes are introduced into the specimen andbecome attached specifically to certain specimen constituents. From thefluorescence properties of the prepared specimen conclusions can bedrawn, for example, as to the nature and composition of the specimen orthe concentration of certain substances within the specimen.

In scanning microscopy in particular, methods that exploitlocation-dependent nonlinearities of the specimen are used. This fieldincludes, for example, coherent anti-Stokes Raman scattering (CARS),which is known inter alia from PCT Application WO 00/04352 A1. It mustbe noted, however, that illuminating light having at least two differentilluminating light wavelengths, at high light power levels, is requiredfor this method.

Another method that makes use of nonlinear effects is so-called STED(stimulated emission depletion) microscopy, known for example fromPCT/DE/95/00124. Here the lateral edge regions of the focus volume ofthe excitation light beam are illuminated with a light beam of anotherwavelength, called the stimulation light beam, that is emitted by asecond laser, so that the specimen regions excited there by the light ofthe first laser are brought back to the ground state in stimulatedfashion. Only the light spontaneously emitted from the regions notilluminated by the second laser is then detected, resulting overall inimproved resolution.

In multi-photon scanning microscopy, the fluorescent photonsattributable to a two-photon or multi-photon excitation process aredetected. The probability of a two-photon transition depends on thesquare of the excitation light power level, and therefore occurs withhigh probability at the focus of the scanning illuminating light beam,since the power density is highest there. To achieve sufficiently highlight power levels, it is useful to pulse the illuminating light andthereby achieve high peak pulsed light power levels. This technique isknown, and is disclosed e.g. in U.S. Pat. No. 5,034,613 “Two-photonlaser microscopy” and in German Unexamined Application DE 44 14 940. Afurther advantage of multi-photon excitation especially in confocalscanning microscopy lies in the improved bleaching behavior, since thespecimen bleaches out only in the region of sufficient power density,i.e. at the focus of an illuminating light beam. Outside that region, incontrast to one-photon excitation, alnost no bleaching takes place.

In scanning microscopy, a specimen is illuminated with a light beam inorder to observe the reflected or fluorescent light emitted from thespecimen. The focus of an illuminating light beam is moved in a specimenplane by means of a controllable beam deflection device, generally bytilting two mirrors, the deflection axes usually being perpendicular toone another so that one mirror deflects in the X direction and the otherin the Y direction. Tilting of the mirrors is brought about, forexample, by means of galvanometer positioning elements. The power levelof the light coming from the specimen is measured as a function of theposition of the scanning beam. The positioning elements are usuallyequipped with sensors to ascertain the present mirror position.

In confocal scanning microscopy specifically, a specimen is scanned inthree dimensions with the focus of a light beam.

A confocal scanning microscope generally comprises a light source, afocusing optical system with which the light of the source is focusedonto an aperture (called the “excitation pinhole”), a beam splitter, abeam deflection device for beam control, a microscope optical system, adetection pinhole, and the detectors for detecting the detected orfluorescent light. The illuminating light is coupled in via a beamsplitter. The fluorescent or reflected light coming from the specimentravels back through the beam deflection device to the beam splitter,passes through it, and is then focused onto the detection pinhole behindwhich the detectors are located. Detection light that does not derivedirectly from the focus region takes a different light path and does notpass through the detection pinhole, so that a point datum is obtainedwhich results, by sequential scanning of the specimen, in athree-dimensional image. A three-dimensional image is usually achievedby acquiring image data in layers, the track of the scanning light beamon or in the specimen ideally describing a meander (scanning one line inthe X direction at a constant Y position, then stopping the X scan andslewing by Y displacement to the next line to be scanned, then scanningthat line in the negative X direction at constant Y position, etc.). Toallow acquisition of image data in layers, the specimen stage or theobjective is displaced after a layer has been scanned, thus bringing thenext layer to be scanned into the focal plane of the objective.

Spectral influencing of light pulses by amplitude modulation or phasemodulation is known from the literature, e.g. from Rev. of ScientificInstruments 71 (5) pp. 1929-1960. Spectral modification of the laserpulses is usually used to shorten the pulses, to shape them optimally,or to control optically induced processes.

The aforesaid methods are disadvantageous in that high light powerlevels are necessary, resulting on the one hand in great demands on thelight source and on the other hand in undesirable damage to thespecimen, for example due to bleaching.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a method formicroscopy that reliably and efficiently allows exploitation ofnonlinear processes with reduced specimen damage.

The invention provides a method comprising the following steps:

-   -   generating pulsed illuminating light that comprises wavelengths        which lie in a spectral region;    -   defining a detection spectral region that lies within the        spectral region;    -   influencing the light components of the illuminating light that        comprise wavelengths within the detection spectral region;    -   illuminating a specimen with the illuminating light;    -   detecting the detection light proceeding from the specimen        within the detection spectral region.

A further object of the invention is to provide a microscope whichreliably and efficiently allows an investigation of a specimenexploiting nonlinear processes with reduced specimen impact.

The invention also provides a microscope having a light source forgenerating pulsed illuminating light that comprises light from aspectral region, and having at least one detector for detecting thedetection light proceeding from a specimen in a detection spectralregion, wherein the detection spectral region lies within the spectralregion; and the illuminating light contains no light from the detectionspectral region having the same polarization properties.

The invention has the advantage that the method according to the presentinvention exploits location-dependent optical nonlinearities but makesdo with much lower light intensities, the use of the lowest possiblelight intensities having particular significance especially forbiological specimens. Investigation of the specimen to a great depth isalso possible.

An aspect of the method according to the present invention is toinfluence, certain spectral components (specifically those from thedetection spectral region) from the spectrum of ultrashort laser pulses(i.e. preferably picosecond and femtosecond laser pulses); to focus theilluminated light prepared in this fashion onto a specimen volume; andto detect in practically background-free fashion the detection lightthereby produced, by nonlinear processes, in the region of thepreviously removed spectral components. The power level and (optionally)spectral distribution of this detection light is used for imageproduction.

In a preferred embodiment, the influencing is a removal of the lightcomponents of the illuminating light that comprise wavelengths withinthe detection spectral region. In another embodiment, the influencingcontains a modification of the polarization state of the lightcomponents of the illuminating light that comprise wavelengths withinthe detection spectral region. The modification of the polarizationstate can encompass, in particular, a rotation of a linear polarization.Rotation of the linear polarization direction makes the detection lightin the detection spectral region distinguishable from the illuminatinglight.

In another preferred embodiment, the influencing encompasses a spectralfiltration. Provided for this purpose, in an embodiment, is a spectralfilter that removes from the illuminating light the light components ofthe illuminating light that comprise wavelengths within the detectionspectral region. In this embodiment, the illuminating light contains nolight from the detection spectral region. In another variant, a spectralfilter is provided that modifies the polarization state of the lightcomponents of the illuminating light that comprise wavelengths withinthe detection spectral region.

The spectral filtration removes certain frequency regions from thespectrum of the illuminating light in order to create there a spectralwindow within which detection light produced as a result of nonlinearprocesses can be detected in background-free fashion.

In a preferred embodiment, a further spectral filter is provided thatallows only light of the wavelengths of the detection spectral region toarrive at the detector. The further spectral filter is preferablyinverse with respect to the spectral filter.

In an embodiment, the illuminating light is already generated in such away that the detection spectral region lies within the spectral region,and so that the illuminating light contains no light from the detectionspectral region. The detection spectral region or regions can, forexample, be the spectral gaps between the equidistant modes of amode-coupled pulsed laser.

In another embodiment, the microscope is a scanning microscope, inparticular a confocal scanning microscope.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the invention is depicted schematically in thedrawings and will be described below with reference to the Figures,identically functioning elements being labeled with the same referencecharacters. In the drawings:

FIG. 1 shows a microscope according to the present invention;

FIG. 2 shows masks for spectral filters;

FIG. 3 shows a further microscope according to the present invention;

FIG. 4 shows a further microscope according to the present invention;

FIG. 5 shows a further microscope according to the present invention;

FIG. 6 shows a further microscope according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 schematically shows a microscope according to the presentinvention that is embodied as a scanning microscope. The opticalcomponents for guiding, directing, and focusing illuminating light beam1 (generated by a pulsed laser 7) and detection light beam 3, and theapparatuses for evaluating the detection light data and displaying animage of the specimen, are not shown in the interest of better clarity.These components are sufficiently familiar to one skilled in the art.

The microscope contains a spectral filter 5 that removes fromilluminating light beam 1 the light components of the illuminating lightthat comprise wavelengths within the detection spectral region. For thatpurpose, the light is spatially spectrally split using a first grating9, and then focused with first lens 11 onto a mask 13 which removes thespectral components that lie within the detection spectral region.Grating 9 and first mask 13 are located in the focal planes of lens 11in a 4f arrangement. Mask 13 has transparent and opaque regions. It canbe expressed as a static mask but also as a dynamically controllablemask (liquid crystal display, hinged mirror array). After first mask 13,the various spectral components of the illuminating light are combinedagain, by means of a symmetrical arrangement of a second lens 15 andsecond grating 17, into a common illuminating light beam 1. Thisilluminating light beam is then coupled into the microscope beam pathand focused by an objective 19 onto specimen 21 that is to be examined.The microscope scans, for example, by the fact that one or more mirrorsin the beam path are embodied as scanning mirrors, and/or by moving thespecimen stage. In the interior of specimen 21 at the location of thefocus of illuminating light beam 1, nonlinear processes such asself-phase modulation, continuum generation, etc. take place, in whichnew light frequencies are generated that may also be present, interalia, in the regions filtered out by the previous stop. After passagethrough the specimen, detection light beam 3 proceeding from specimen 21is collimated by a condenser 23 and directed to a further spectralfilter 25. Further spectral filter 25 is embodied inversely with respectto spectral filter 5 through which illuminating light beam 1 passes;i.e. wherever light previously passed through, the light is now blocked.It contains a third lens 29 and a fourth lens 31, as well as a thirdgrating 33 and a fourth grating 35; also a second mask 37 that is theinverse of first mask 13. The components of illuminating light beam 1still present in detection light beam 3 are thereby filtered out, sothat ultimately only detection light produced at the specimen focusarrives at detector 27. The power level of this light providesinformation, inter alia, about the nonlinear refractive indices at thespecimen focus which depend on local conditions in specimen 21, and istherefore suitable, as the focus is scanned over specimen 21, as asignal for image-producing methods.

FIG. 2 shows several spatial first masks 13 and second masks 37 that canbe used in first spectral filter 5 and in second spectral filter 25,second masks 37 being inverse with respect to first masks 13. Thetransmitting regions can be limited even further.

FIG. 3 shows a further microscope according to the present invention. Itcorresponds analogously, in terms of illumination, to the scanningmicroscope shown in FIG. 1; several detectors 39, 41, 43, 45 arrangedbehind second mask 37 are provided for detection. A linear detector oran array of detectors (e.g. CCD) could also be used. After spectralsplitting using grating 33, the components of detection light beam 3that comprise the same wavelength region as the components ofilluminating light beam 1 that were removed by first mask 13 strike theseveral individual detectors 39, 41, 43, 45. In a particularly simplearrangement, the mask itself can even be omitted. Because the severaldetectors 39, 41, 43, 45 are used, additional information is obtained asto the intensity with which the nonlinear processes are occurring in thevarious spectral regions; this can possibly be utilized fordifferentiated image production.

It is also possible to use for second spectral filter 25 at least someof the same optical elements as for first spectral filter 13, by guidingthe light beam through at least some of them a second time.

FIG. 4 shows a further microscope according to the present invention.Instead of gratings 9, 17, 33, 35, a first prism 47, second prism 49,third prism 51, and fourth prism 53 are used for spectral splitting andcombining. Illuminating light beam 1 generated by pulsed laser 7 islinearly polarized. Mask 13 rotates through 90 degrees the polarizationdirection of those components of illuminating light beam 1 that comprisewavelengths from the detection regions. The polarization influence isexerted by way of a suitably patterned and oriented birefringent fixedmask 13 (e.g. patterned λ/2 plate), or also by means of a dynamicallycontrolled mask 13 that can be implemented, for example, using a liquidcrystal display. After passage through the specimen, the detection lightproceeding from the specimen is filtered through a second spectralfilter 25 in such a way that the illuminating light whose polarizationwas not rotated by first spectral filter 5 is completely removed. Thisis done by the fact that in second spectral filter 25, by way of asuitable second mask 37, the polarization state of the various spectralcomponents is modified in such a way that all components derivingdirectly from pulsed laser 7 are once again given a common polarization,which is removed from the beam path by means of a downstream polarizer55. In the concrete exemplary embodiment, those spectral components thathad already experienced a polarization change in first spectral filter13 are once again rotated 90° in polarization in second spectral filter25. Polarizer 55 then removes from the beam all spectral components thathave a polarization of 0°. The beam path then, as a rule, contains onlylight which was produced in the specimen by nonlinear processes, andwhose intensity permits conclusions as to the local nonlinear refractiveindices of the specimen at the focus and is therefore suitable for imageproduction. In this exemplary embodiment it is also possible to dispensewith certain parts of second spectral filter 25 (e.g. fourth lens 31 andfourth prism 53) if, for example, the detector(s) is/are equipped withpolarizers and is/are arranged directly behind mask plane 25.

FIG. 5 shows an embodiment in which the light polarized in the 0° or 90°direction (depending on wavelength) is split upstream from the specimenusing a polarization splitter 57, after which the two light componentsof illuminating light beam 1 are focused from opposite directions ontospecimen 21 by a first objective 59 and a further objective 61. Here theobjective for the one polarization direction is in each casesimultaneously the condenser for the other polarization direction. Afterpassage through the specimen and through a polarization rotator 62 (thisnumber has already been assigned to the second objective, including inthe Figure), which is embodied as a λ/2 plate 63 that preferably rotatesthe polarization 90°, the two light components of the detection lightare combined using the polarizing beam splitter; the light uninfluencedby the specimen is separated, by polarizing beam splitter 57, from thelight later to be detected in such a way that only the light justproduced in the specimen is detected in detector 27.

In the exemplary embodiment in FIG. 6, the first spectral filter hasbeen omitted. The light of pulsed laser 7 is made up of lines lying veryclose together. This occurs in many usual picosecond and femtosecondlasers as an effect of mode coupling. The spectral line spacing usuallycorresponds here to the pulse frequency of the laser in question; forexample, the spectrum of a titanium-sapphire femtosecond laser pulsingat a repetition rate of 80 MHz is made up of individual spectral linesat a spectral spacing of 80 MHz. Gaps in the spectrum are presentbetween the individual spectral lines, so that the spectrum of thispulsed laser 7 is similar to the filtered spectra of the exemplaryembodiments discussed previously. If components are present in thesespectral regions after an excitation laser of this kind has passedthrough specimen 21, this is attributable to nonlinear processes; as inthe case of the previous exemplary embodiments, this can be utilized forimage production. Separation of the detection light produced bynonlinear processes from the excitation light can be accomplished, as inthe previous exemplary embodiments, by spatial filtration; in thiscontext, the use of monochromators, etc. of course also represents aspatial filtration. Alternatively and in particularly preferred fashion,what is used as second spectral filter 25 is an etalon 63, which isconstituted by a first mirror 65 and a second mirror 67 and whichremoves from detection light beam 3 all spectral components within acertain wavelength spacing (as is also possible, in the previousexemplary embodiments, with a suitable first spectral filter). In thecase of the mode-coupled laser, the spectral distance within whichetalon 63 absorbs light must correspond exactly to the spectral spacingof the individual laser modes, which substantially means that the lengthof etalon 63 must be matched to the effective resonator length of themode-coupled laser. Since the etalon length is relatively long for theshort-pulse lasers commonly in use at present, etalon 63 is usuallyembodied as a resonator made up substantially of two semitransparentmirrors 65, 67 at the spacing of the effective resonator length. It isuseful if there is located, in the interior of the resonator, acontrollable element 69 with which the effective resonator length can beregulated so that precise adaptation can be performed and with which anydrift resulting e.g. from thermal longitudinal expansion can becontrolled out. An element of this kind could be made of materials whoserefractive index can be controlled externally, e.g. liquid crystals orferroelectric crystals. Appropriate regulation of the etalon's resonatorlength could also be accomplished by way of a movable end mirror.Instead of the resonator length of the etalon, the length of theshort-pulse laser resonator could, of course, also be regulated.

The invention has been described with reference to exemplaryembodiments. It is self-evident, however, that changes and modificationscan be made without thereby leaving the range of protection of theclaims below.

1. A method for microscopy comprising: generating pulsed illuminatinglight including wavelengths in a spectral region; defining a detectionspectral region within the spectral region; influencing, using adynamically controllable mask, light components of the illuminatinglight that comprise wavelengths within the detection spectral region;illuminating a specimen with the influenced illuminating light; anddetecting detection light proceeding from the specimen within thedetection spectral region.
 2. The method as defined in claim 1, whereinthe dynamically controllable mask includes at least one of a liquidcrystal display and a hinged mirror array.
 3. The method as defined inclaim 1, wherein the influencing includes a removal of the lightcomponents of the illuminating light that comprise wavelengths withinthe detection spectral region.
 4. The method as defined in claim 1,wherein the influencing includes a modification of the polarizationstate of the light components of the illuminating light that comprisewavelengths within the detection spectral region.
 5. The method asdefined in claim 4, wherein the modification of the polarization stateencompasses a rotation of a linear polarization.
 6. The method asdefined in claim 4, further comprising modifying the polarization stateof light components of the detection light.
 7. The method as defined inclaim 1, wherein the influencing encompasses a spectral filtration. 8.The method as defined in claim 1, wherein a pulsed laser is provided forgenerating the pulsed illuminating light.
 9. The method as defined inclaim 1 further comprising allowing, using a further spectral filter,only light of wavelengths of the detection spectral region to arrive atthe detector, wherein further spectral filter is at least partiallyinverse with respect to the spectral filter.
 10. A microscopecomprising: a light source configured to generate pulsed illuminatinglight that includes light from a spectral region; at least one detectorconfigured to detect detection light proceeding from a specimen in adetection spectral region, the detection spectral region being withinthe spectral region; and a spectral filter including a dynamicallycontrollable mask configured to influence light components of theilluminating light that comprise wavelengths within the detectionspectral region.
 11. The microscope as defined in claim 10, wherein thedynamically controllable mask includes at least one of a liquid crystaldisplay and a hinged mirror array.
 12. The method as defined in claim10, wherein the dynamically controllable mask is configured to removethe light components of the illuminating light that comprise wavelengthswithin the detection spectral region.
 13. The method as defined in claim10, wherein the dynamically controllable mask is configured to modifythe polarization state of the light components of the illuminating lightthat comprise wavelengths within the detection spectral region.
 14. Themethod as defined in claim 13, wherein the modification of thepolarization state encompasses a rotation of a linear polarization. 15.The method as defined in claim 13, further comprising a further spectralfilter configured to modify the polarization state of light componentsof the detection light.
 16. The microscope as defined in claim 10,further comprising a further spectral filter configured to allow onlylight of wavelengths of the detection spectral region to arrive at thedetector, wherein the further spectral filter is at least partiallyinverse with respect to the spectral filter.
 17. The microscope asdefined in claim 10, wherein the light source includes a pulsed laser.18. A microscope comprising: a light source configured to generatepulsed illuminating light that includes light from a spectral region; atleast one detector configured to detect detection light proceeding froma specimen in a detection spectral region, the detection spectral regionbeing within the spectral region; a spectral filter configured toremove, from the illuminating light, light components of theilluminating light that comprise wavelengths within the detectionspectral region; and a further spectral filter configured to allow onlylight of wavelengths of the detection spectral region to arrive at thedetector, wherein the further spectral filter is at least partiallyinverse with respect to the spectral filter.
 19. The microscope asdefined in claim 18, further comprising a third spectral filterconfigured to modify the polarization state of the light components ofthe illuminating light that comprise wavelengths within the detectionspectral region.
 20. The method as defined in claim 18 wherein the lightsource includes a pulsed laser.